Antifungal therapy in the management of fungal secondary infections in COVID-19 patients: A systematic review and meta-analysis

Objectives The prevalence of fungal secondary infections among COVID-19 patients and efficacy of antifungal therapy used in such patients is still unknown. Hence, we conducted this study to find the prevalence of fungal secondary infections among COVID-19 patients and patient outcomes in terms of recovery or all-cause mortality following antifungal therapy (AFT) in such patients. Methods We performed a comprehensive literature search in PubMed®, Scopus®, Web of Sciences™, The Cochrane Library, ClinicalTrial.gov, MedRxiv.org, bioRxiv.org, and Google scholar to identify the literature that used antifungal therapy for the management fungal secondary infections in COVID-19 patients. We included case reports, case series, prospective & retrospective studies, and clinical trials. Mantel Haenszel random-effect model was used for estimating pooled risk ratio for required outcomes. Results A total of 33 case reports, 3 case series, and 21 cohort studies were selected for final data extraction and analysis. The prevalence of fungal secondary infections among COVID-19 patients was 28.2%. Azoles were the most commonly (65.1%) prescribed AFT. Study shows that high survival frequency among patients using AFT, received combination AFT and AFT used for >28 days. The meta-analysis showed, no significant difference in all-cause mortality between patients who received AFT and without AFT (p = 0.17), between types of AFT (p = 0.85) and the duration of AFT (p = 0.67). Conclusion The prevalence of fungal secondary infections among COVID-19 patients was 28.2%. The survival frequency was high among patients who used AFT for fungal secondary infections, received combination AFT and AFT used for >28 days. However, meta-analysis results found that all-cause mortality in COVID-19 patients with fungal secondary infections is not significantly associated with type and duration of AFT, mostly due to presence of confounding factors such as small number of events, delay in diagnosis of fungal secondary infections, presence of other co-infections and multiple comorbidities.


Introduction
Severely ill coronavirus disease-19  patients, admitted to intensive care units (ICUs) are at increased risk of bacterial and fungal secondary infections. Pulmonary aspergillosis, invasive candidiasis, and mucormycosis are the most frequently reported fungal secondary infections, leading to increased morbidity and mortality in COVID-19 patients [1]. The most common pathogens reported belongs to Aspergillus, Rhizopus and Candida species. Looking back to 2003, the incidence of fungal secondary infections in COVID-19 patients was high and ranges from 14.8-33% in mild to severely ill patients [2]. However, the recent clinical scenarios from the globe have raised concerns about fungal secondary infections and their management in COVID-19 patients [3]. The presence of diabetes mellitus, cancers, additional immunocompromised status, use of steroids and/or immunosuppressive agents, use of mechanical ventilators are some of the identified risk factors for fungal secondary infections in hospitalized COVID-19 patients [4]. The time interval between COVID-19 diagnosis and the development of fungal co-infection varies widely. In addition, the abrupt development of clinical features makes it more fatal [5]. Therefore, early detection and management help to prevent severe illness and associated deaths.
Currently, there are no established guidelines for the management of fungal secondary infections in COVID-19 patients. However, there are various case reports, case series, and cohort studies published with regard to the management of fungal secondary infections in COVID-19 patients. The commonly used antifungal therapy (AFT) includes liposomal amphotericin B, azole, and echinocandins [6]. Hence, we conducted this study to systematically published literatures to explore the prevalence of fungal secondary infections among COVID-19 patients and outcomes in terms of recovery or all-cause mortality associated with the use of AFT in such patients.
antifungal agent was used. The fungal secondary infections were defined as those caused by any fungal species either at admission or during the hospital stay. The fungal species were detected by observing the colony morphology and color of the isolated culture media. The details of inclusion criteria are presented in PICOS format in S1 Appendix. We excluded review articles, systematic reviews, meta-analysis, brief reports, short reports, editorials, commentaries, notes, book chapters, abstracts, surveys, conference proceedings, posters presentations, unpublished materials and guidelines.

Data sources
We performed a comprehensive literature search using predefined search terms in eight online search engines namely, PubMed 1 , Scopus 1 , Web of Sciences™, The Cochrane Library (Central), ClinicalTrial.gov, MedRxiv.org, bioRxiv, and Google scholar to identify the literature records published between 1 st January 2020 and 30 th June 2021. A manual hand search of references was also performed to avoid missing any relevant literature. Further, all the literatures retrieved from the search engines were transferred to the Mendeley reference manager to remove duplicate records. The details of search strategies are presented in S2 Appendix.

Study selection
The study titles and abstracts were independently screened by two authors to determine whether the studies met the inclusion criteria. The full-text records of these studies were further reviewed for final inclusion. Additionally, the reference section of all the selected articles were hand-searched by another author, to identify the additional literature records for possible inclusions. If any missing study relevant information, review authors were actively participated in the searching for original resources or contacted study authors through mail to obtain missing information. The discrepancies related to the selection and eligibility were resolved through discussion between the first three authors, and unresolved issues were addressed by the 4 th and 5 th authors. The final decision was made following consensus between all the authors.

Data extraction
Two authors independently performed the data extraction from all the included records and were documented in a specifically designed data extraction tool (©Microsoft excel-2019). The variables such as the first author of the publication, year of publication, geographical location where the study was performed, type of the study (case reports, case series, prospective studies, retrospective studies and clinical trials), sample size, age (in year) and gender, diagnosis of fungal co-infection, types of fungal species isolated / cultured, name of antifungal drugs, type of therapy (mono or combination), dose, frequency & route of administration, total duration of antifungal therapy (in days), total duration of hospital stay (in days), and patient outcomes (either alive or dead) were recorded.

Data synthesis
The outcome measures were to assess the prevalence of fungal secondary infections (cohort studies), all-cause mortality in patients using AFT and without AFT, all-cause mortality associated with type of AFT (mono or combination AFT), and all-cause mortality associated with the duration of AFT (�28 days or >28 days) among COVID-19 patients with fungal secondary infections.

Statistical analysis
A meta-analysis was performed for all the eligible cohort studies. If three or more studies reporting any or similar fungal secondary infections and use of AFT were identified, and applied Mantel Haenszel random-effect model for estimating pooled risk-ratio using Review Manager (RevMan) 5.4.1 software ([Computer program] Version 5.3. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014) for required outcomes. I 2 statistic was used to evaluate the heterogenicity of studies following Cochrane recommendations [9] and heterogenicity was considered substantial if I 2 was >50%.

Risk of bias assessment
We used the methodological quality & synthesis guide for evaluating the risk of bias involved in case reports, and case series. Based on the total score, the methodological quality & synthesis guide categorizes the risk of bias as low (5), medium (3)(4) and high (0-2) [10]. Whereas, Newcastle-Ottawa Quality scale was used to assess the quality of the cohort studies [11]. A total score of three or less is indicative of poor quality, 4-6 as moderate quality, and 7-9 as high quality of cohort studies [12].

Study selection
A total of 403 records were identified in the scientific databases and hand search of references. After removing the 102 duplicate records, the remaining 301 records were screened for title and abstract. Of them, 162 records were excluded as they were irrelevant to the study. Further, 139 full text articles were reviewed and 82 articles were excluded from them (S1 Table). Finally, 33 case reports [18,, 3 case series [55][56][57] and 21 cohort studies  were selected for final data extraction and analysis (Fig 1).

PLOS ONE
Antifungal therapy in fungal secondary infections in COVID-19 patients: A systematic review and meta-analysis

PLOS ONE
Antifungal therapy in fungal secondary infections in COVID-19 patients: A systematic review and meta-analysis

PLOS ONE
Antifungal therapy in fungal secondary infections in COVID-19 patients: A systematic review and meta-analysis

Duration of Antifungal Therapy (AFT) and hospital stay
3.6.1. Case report and case series. Among 38 patients in case reports, a majority (n = 21, 55.2%) received AFT for �28 days, 11 patients (28.9%) for >28 days, and 6 patients (15.7%) the duration of AFT was not adequately reported. The total duration of hospital stays among the study patients ranged from 2 to 175 days. During hospital stay, the majority of patients were received ICU care or mechanical ventilation support. (Table 1).
Three studies [62,63,68] were included in meta-analysis and found that the frequency of all-cause mortality was lower among patients who received combination AFT [7/13, 53.8%] as compared to the patients using monotherapy [9/14, 64.2%] for the management of fungal secondary infections. However, the pooled risk ratio showed that there was no significant difference in all-cause mortality between these groups [RR: 1.08, 95% CI: 0.48-2.43, p = 0.85, I 2 = 39%] [Fig 2(B)].
Further, we included three studies [65,70,71] in meta-analysis to assess the all-cause mortality among CAPA patients with AFT and without AFT. Though the frequency of all-cause mortality was lower among CAPA patients who were on AFT [12/22, 54.5%] as compared to those who weren't on AFT [5/8, 62.5%], the pooled risk ratio revealed no significant difference between the groups [RR: 0.74, 95% CI: 0.40-1.36, p = 0.33, I 2 = 17%] [Fig 2(D)]. The metaanalysis of three studies [70][71][72] that assessed the association between the all-cause mortality and duration of AFT (for �28 days vs >28 days) among CAPA patients showed that the frequency of all-cause mortality was lower in patients who were on AFT for >28 days as compared to those used AFT for �28 days. However, the pooled risk-ratio revealed no significant difference between the groups [RR: 1.47, 95% CI: 0.57-3.79, p = 0.43, I 2 = 32%] [Fig 2(E)].
The summary of fungal secondary infections, AFT used, duration of AFT, and outcomes among COVID-19 patients is presented in S2 Table.

Discussion
The worldwide mortality associated with COVID-19 is 3.97 million [13]. However, there is no published literature suggesting the global mortality in COVID-19 patients with fungal secondary infections. In this review, the prevalence of fungal secondary infections (cohort studies) was 28 [2/15] respectively. The mortality rate associated with CAPA was lower (51.2%) in a recent review published by Singh S et al., [14] another recent review reported the mortality associated with CAM as 30.7% [15]. The mortality associated with CAC was not adequately reported in the studies that we reviewed, however a recently published study from Atlanta reported that CAC associated mortality was up to 30.9% [16]. Thus, the overall mortality rates associated with fungal secondary infections in COVID-19 patients are higher as compared to COVID-19 alone. There could be various contributing factors for this such as type of fungal species, multiple fungal secondary infections, AFT used, presence of other bacterial or viral superinfections, use of immunosuppressive therapy, presence of other co-morbid conditions, and age of the patients. CAPA (n = 36 studies) [23-35, 37-42, 55-58, 61, 63-65, 67, 69-75, 77] was the most commonly diagnosed fungal co-infection, followed by CAC (n = 9 studies) [49,58,60,66,68,69,74,76,77] and CAM (n = 9 studies) [18, 43-48, 59, 69]. Many studies from Europe, Australia and China have reported increased prevalence of CAPA (range: 20-35%) [17]. We observed that most of the studies including patients with CAPA (n = 22 studies) and CAC (n = 5 studies) were reported from Europe. Aspergillus Fumigatus was the most common causative organism identified through culture media in these studies. Voriconazole is the recommended first-line antifungal therapy whereas, amphotericin B is the second-line agent for CAPA [17]. We observed that the most common AFT prescribed for CAPA was voriconazole followed by amphotericin B. The recommended maintenance doses of voriconazole and amphotericin B are 200 mg bid [18] and 3mg/kg/day respectively [19]. In our review, the prescribed dose range of voriconazole was 200 to 800 mg/day and amphotericin B was 3 to 10 mg/kg/day (either as single-drug therapy or as combination). The recommended median duration of AFT is 76 days [18], our review explored that the total duration of AFT ranged from 2 to 90 days.
The prevalence of CAC was high among the Chinese population (23.5%) [20]. The recommended AFT includes echinocandins, azoles and amphotericin B [21]. In this review, we observed that the common causative organism of CAC was Candida albicans and the most common AFT prescribed for CAC was fluconazole followed by nystatin. Fluconazole was prescribed at a dose of 400 mg/day, and the total duration of AFT ranged from 4 to 21 days.
We observed most of the published literature for CAM were from India (n = 5 studies) [18,43,46,48,59]. The most common AFT used for the management of CAM was amphotericin B either as a single drug or in combination with other antifungal drugs. The current guideline for the management of mucormycosis recommends liposomal amphotericin B and posaconazole as the first-line AFT [2]. The recommended dose of amphotericin B is 5 to 10 mg/kg/day [22], we observed that in the reviewed studies amphotericin B was used in the dose range of 3 to 5 mg/kg/day. The total duration of AFT for CAM was ranged from 11 to 150 days.
There were 33 studies that included only CAPA patients [23-42, 55-57, 61, 63-65, 67, 70-73, 75]. Among them 17 studies used single-drug therapy, eleven studies used combination AFT, three studies used both single-drug and combination AFT, and two studies used AFT the details of which were not adequately reported.
Among nine studies [18, 43-48, 59, 60] that reported only CAM, two studies used liposomal amphotericin B alone, five studies used combination AFT (liposomal amphotericin B with azoles, liposomal amphotericin B with caspofungin and azole, voriconazole and caspofungin) and one study used both single-drug as well as combination AFT. It was observed that all the patients in the studies, where single-drug therapy was used were alive. Among seventeen patients that received combination therapy, five were dead and remaining were alive.
There were five studies [49,54,60,66,68] that included only CAC patients. Two studies used single-drug therapy, two studies used both single-drug as well as combination AFT and one study used AFT the details of which are not adequately reported. In two studies where single-drug therapy was used all the patients were alive, whereas in other studies the mortality details were not adequately reported.
The results of meta-analysis revealed that there was no significant difference in terms of allcause mortality among patients who received AFT & did not receive AFT (p = 0.17), all-cause mortality & type of AFT used (p = 0.85), and all-cause mortality & duration of AFT (p = 0.87). There could be various confounding factors such as delay in diagnosis of fungal secondary infections in earlier or terminal stages of COVID-19 by physicians, diagnostic difficulties in mycological detection, increased risk of bacterial or viral infections in short to long term of infections, presence of polymorbidity and low sample size might be the reasons for the nonsignificance differences found in all-cause mortality with who received & did not receive AFT, type of AFT used and duration of AFT.
Further, the sub-group analysis including studies that reported CAPA patients alone, revealed that was no significant difference in terms of all-cause mortality among patients who received AFT & did not receive AFT (p = 0.33), and all-cause mortality & duration of AFT (p = 0.43). However, in CAPA patients also, we observed a high survival frequency among patients who used AFT [10/22, 45.4%] and when AFT was used for >28 days [5/8, 62.5%]. However, we couldn't find the studies for similar subgroup meta-analysis in patients with CAC and CAM.
At the time of literature search, there was no published literature available on randomized control studies conducted among patients with fungal secondary infections associated with COVID-19. However, we made an attempt to explore if there are any such ongoing studies. Our search revealed that currently there are only two ongoing studies. One of which is phase 2 and another one is phase 3 study. The expected date of completion of these studies will be first quarter of 2022. The availability of these study results will hopefully add to the existing evidence of efficacy of AFT in treating fungal secondary infections among COVID-19 patients. The details of these ongoing studies are presented in S5 Table.

Limitations
We could not able to establish the efficacy of any individual AFT or class of antifungal agent/s that are used for the treatment of fungal secondary infections in COVID-19 patients due to a lack of adequate data reporting among the included studies about antifungal regimen. It was observed that many studies were reported antifungal drugs without complete information about antifungal regimens including doses, frequency and duration. In addition, at the time of literature search, there was no published literature on randomized control studies conducted in COVID-19 patients with fungal secondary infections. Availability of such literature would have added more clarity on efficacy of AFT in different fungal secondary infections associated with COVID-19 patients.

Conclusion
The prevalence of fungal secondary infections among COVID-19 patients was 28.2%. The most common fungal secondary infections among COVID-19 patients were CAPA, CAC and CAM. Voriconazole, fluconazole and liposomal amphotericin B were the most commonly used antifungal agents for the management of CAPA, CAC and CAM respectively. The results of this systematic review and meta-analysis suggest that the survival frequency was high among patients who were; on AFT for the management of fungal secondary infections, using combination AFT and using AFT for >28 days. However, the pooled risk ratio, revealed that the all-cause mortality in COVID-19 patients with fungal secondary infections is not associated with the type and duration of AFT may be due to the availability of confounding factors such as delay in diagnosis of fungal secondary infections, presented with multiple comorbidities, older age and a small number of events that may reduced power to detect a difference, may contribute for outcomes in such patients.